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1.
A new fluorogenic compound--6-hexadecanoylamino-4-methyl-umbelliferyl-beta-D-gala cto pyranoside (HMGal), a substrate for human galactocerebroside beta-D-galactosidase (HG), has been synthesized. A method for determining the HG activity based on the use of HMGal as a fluorogenic substrate has been developed. The specificity of HMGal hydrolysis by HG has been demonstrated in experiments with enzyme preparations from human skin fibroblasts and leukocytes in normally and in hereditary glycolipidosis (GM1-gangliosidosis and Krabbe's disease). The use of HMGal permits to markedly increase the sensitivity of the method used for determining the HG activity.  相似文献   

2.
Free sialic acid has been found in the cell-conditioned medium of human foreskin fibroblasts. It is proposed that the accumulation of extracellular sialic acid may result from the hydrolysis of GM3 ganglioside on the cell surface of these fibroblasts. Sialidase activities with GM3 ganglioside and sialyllactitol as substrates were demonstrated in cell-conditioned medium, and the levels of their activities correlated positively with cell density. The GM3 sialidase activity at pH 4.5 was 4.1 and 38 pmol/h/ml of medium at sparse and confluent densities, respectively; the corresponding activities with sialyllactitol as the substrate were 12 and 75 pmol/h/ml of medium (pH 4.5). The pH versus activity profiles with GM3 as the substrate suggested the presence of a second sialidase with an optimal activity at pH 6.5 in the conditioned medium of preconfluent cells. This activity was virtually absent in the medium of contact-inhibited cells and could not be assayed with sialyllactitol as the substrate. The turnover of cell surface GM3 was assessed by pulse labeling human foreskin fibroblasts with a radioactive precursor of sialic acid ([1-14C]N-acetylmannosamine) and a radioactive precursor of ceramide ([3,3-3H2]serine). During a chase period of 24 h turnover of the doubly labeled cellular GM3 was observed; there was a loss of about 35% of the 14C-labeled sialic acid without any measureable loss of 3H-labeled ceramide from GM3. We have speculated that the enzyme-catalyzed removal of sialic acid from the GM3 ganglioside on the extracellular aspect of the plasma membrane may be a necessary event involved in the modulation of cell growth.  相似文献   

3.
The ability of phospholipase A2 from porcine pancreas to degrade all of the available dilauroylphosphatidylcholine in mixed monolayers with galactocerebroside, sulfatide, or ganglioside GM1 was investigated at different constant surface pressures. Under the conditions used the interfacial glycosphingolipid composition was continuously enriched as the enzyme action proceeded. The total percentage of phospholipid degradation depends on the surface pressure and on the type of glycosphingolipid. The presence of sulfatide activates the enzyme while galactocerebroside and ganglioside GM1 are inhibitory. The extent of phospholipid hydrolysis is independent of the effect of glycosphingolipids on the enzyme velocity. This is so when the latter is measured either in conditions of constant glycosphingolipid composition and zero-order kinetics [Bianco, I.D., Fidelio, G.D., & Maggio, B. (1989) Biochem. J. 258, 95-99] or under variable surface composition as in the present work. The modulation of phospholipase A2 activity by glycosphingolipids operates at two independent levels. One controls the rate of enzyme activity, and the other modulates the total extent of substrate degradation. This depends on the initial interaction of the enzyme with the interface. The glycosphingolipid effect on the activity is different depending on whether the enzyme has access to the substrate from the subphase or is already adsorbed to the lipid interface.  相似文献   

4.
A neuraminidase activity in myelin isolated from adult rat brains was examined. The enzyme activity in myelin was first compared with that in microsomes using N-acetylneuramin(alpha 2----3)lactitol (NL) as a substrate. In contrast to the microsomal neuraminidase which exhibited a sharp pH dependency for its activity, the myelin enzyme gave a very shallow pH activity curve over a range between 3.6 and 5.9. The myelin enzyme was more stable to heat denaturation (65 degrees C) than the microsomal enzyme. Inhibition studies with a competitive inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, showed the Ki value for the myelin neuraminidase to be about one-fifth of that for the microsomal enzyme (1.3 X 10(-6) M versus 6.3 X 10(-6) M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 X 10(-4) M and 4.3 X 10(-4) M, respectively. An enzyme preparation that was practically devoid of myelin lipids was then prepared and its substrate specificity examined. The "delipidated enzyme" could hydrolyze fetuin, NL, and ganglioside substrates, including GM1 and GM2. When the delipidated enzyme was exposed to high temperature (55 degrees C) or low pH (pH 2.54), the neuraminidase activities toward NL and GM3 decreased at nearly the same rate. Both fetuin and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid inhibited NL and GM3 hydrolysis. With 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, inhibition of NL was greater than that of GM3; however, the Ki values for each substrate were almost identical. GM3 and GM1 also competitively inhibited the hydrolysis of NL and NL similarly inhibited GM3 hydrolysis by the enzyme. These results indicate that rat brain myelin has intrinsic neuraminidase activities toward nonganglioside as well as ganglioside substrates, and that these two enzyme activities are likely catalyzed by a single enzyme entity.  相似文献   

5.
The kinetic parameters (Km and V) of human arylsulphatase B (4-sulpho-N-acetylgalactosamine sulphatase) activity in cultured skin fibroblasts were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo chondroitin 4-sulphate and dermatan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, were desulphated up to 4400 times faster than the minimum arylsulphatase-B-specific substrate, namely the monosaccharide N-acetylgalactosamine 4-sulphate. Aglycone structures that influence substrate binding and/or enzyme activity were an adjacent-residue C-6 carboxy group and a second but internal N-acetylgalactosamine 4-sulphate residue. Arylsulphatase B activity in fibroblast homogenates assayed with O-(beta-N-acetylgalactosamine 4-sulphate)-(1----4)-O-D-(beta-glucuronic acid)-(1----3)-O-D-N-acetyl[1-3H] galactosaminitol 4-sulphate derived from chondroitin 4-sulphate as substrate clearly distinguished Maroteaux-Lamy-syndrome patients from normal controls and other mucopolysaccharidosis patients. We recommend the use of the above trisaccharide substrate for both postnatal and prenatal diagnosis of Maroteaux-Lamy syndrome.  相似文献   

6.
Normal quantities of GM1 beta-galactosidase cross reacting material (CRM) (0.31-0.47 microgram/mg protein) were detected by a sensitive radial immunodiffusion assay in skin fibroblasts from patients with GM1 gangliosidosis type 1 and adult variants, whereas elevated levels were found in GM1 gangliosidosis type 2 (0.41-0.72 microgram/mg protein). The specific activity of the immunologically CRM towards GM1 ganglioside of normal fibroblasts was about 500 times that of type 1, 100 times that of type 2, and 30 times that of the adult variants.  相似文献   

7.
Sialidase assays were carried out with the substrate, ganglioside GD1a, coated onto enzyme immunoassay plate wells. Following the incubation of GD1a with sialidase from V. cholerae, the amount of ganglioside GM1 produced was measured as follows: cholera toxin B subunit conjugated to horseradish peroxidase was added to specifically bind to GM1, and then the amount of bound peroxidase was determined in a colorimetric enzymatic assay. In the absence of detergent, linearity for the detection of GM1 was 0 to 0.5 pmol per well, and the sensitivity for sialidase detection was about 3 fmol of product formed per minute. The addition of detergent (Triton CF-54) to the assay reduced the sensitivity and increased the amount of substrate required. Application of this assay for the detection of cell-derived neutral (pH 6.5) sialidase activities in the conditioned medium of human skin fibroblasts is described.  相似文献   

8.
The subcellular distribution of acid carboxypeptidase was investigated in rat liver, normal human skin (CRL 1501) and lung (WI-38) fibroblasts, galactosialidosis skin fibroblasts (GM 00806) and transformed lung fibroblasts (WI-38 VA 13). Results of differential and isopycnic centrifugations and osmotic activation experiments clearly indicate that the enzyme is located in lysosomes, in agreement with observations suggesting that carboxypeptidase is the protective protein of the 'Galjaard complex' which is defective in galactosialidosis.  相似文献   

9.
The effect of neutral (galactocerebroside and asialo-ganglioside GM1) or anionic (sulphatide and gangliosides GM1, GD1a and GT1b) glycosphingolipids on the activity of phospholipase A2 from pig pancreas was studied in mixed monolayers of dilauroyl phosphatidylcholine with the glycosphingolipids in different molar fractions at various constant surface pressures. The activity of the enzyme depends on the proportion and type of glycosphingolipid in the interface. Sulphatide activates the enzyme at all proportions, whereas galactocerebroside shows inhibition or activation depending on its proportion in the film. Asialo-ganglioside GM1 and gangliosides GM1, GD1a and GT1b can strongly inhibit the enzyme at relatively low molar fractions in the film in the following order: asialo-ganglioside GM1 less than ganglioside GM1 less than ganglioside GT1b less than ganglioside GD1a. The changes of activity are not due to a direct action of the lipids on the active centre or interfacial recognition region of the enzyme.  相似文献   

10.
Precursors of cathepsin D and beta-hexosaminidase were isolated from secretions of human fibroblasts and their activity was studied with natural substrates. The immunoprecipitated precursor of cathepsin D, Mr 53000, was inactive with radioactive hemoglobin as substrate. At pH 3.8-4.2 an activation of the precursor took place, which was correlated by a reduction in size to Mr 51500. The observed cleavage of cathepsin D precursor in vitro resembles the autocatalytic activation of pepsinogen. The precursor of beta-hexosaminidase A is able to cleave the natural substrate GM2 ganglioside. This reaction, like that of the mature enzyme, depends on the presence of a protein activator, which interacts with the substrate and the enzyme.  相似文献   

11.
Ganglioside GD1a-GalNAc was isolated from Tay-Sachs brain, tritium-labeled in its sphingosine moiety, and its enzymic degradation studied in vitro and in cultured fibroblasts. When offered as micelles, GD1a-GalNAc was almost not hydrolyzed by Hex A or Hex B, while after incorporation of the ganglioside into the outer leaflet of liposomes, the terminal GalNAc residue was rapidly split off by Hex a. In striking contrast to ganglioside GM2, the major glycolipid substrate of Hex A, the enzymic hydrolysis of GD1a-GalNAc was not promoted by the GM2 activator protein, although the activator protein did bind GD1a-GalNAc to form a water-soluble complex. Pathobiochemical studies corroborate these results. After incorporation of [3H]GD1a-GalNAc into cultured skin fibroblasts from healthy subjects and from patients with different variants of GM2 gangliosidosis, its degradation was found to be strongly attenuated in mutant cells with Hex A deficiencies such as variant B (Tay-Sachs disease), variant B1 and variant 0 (Sandhoff disease), while in cells with variant AB (GM2 activator deficiency), its catabolism was blocked only at the level of GM2. In line with these metabolic studies, a normal content of GD1a-GalNAc was found in brains of patients who had succumbed to variant AB of GM2 gangliosidosis whereas in brains from variants B, B1, and 0, its concentration was considerably elevated (up to 19-fold). Together with studies on the enzymic degradation of GM2 derivatives with modifications in the ceramide portion, these results indicate that mainly steric hindrance by adjacent lipid molecules impedes the access of Hex A to membrane-bound GM2 (whose degradation therefore depends on solubilization by the GM2 activator) and in addition that the interaction between the GM2. GM2 activator complex and the enzyme must be highly specific.  相似文献   

12.
Monogalactosyl diglyceride has previously been demonstrated to be intimately associated with brain white matter, especially myelin. Enzymes responsible for its biosynthesis and degradation have been reported to be present in rat and mouse brain. In the present study, the β-galactosidase responsible for the degradation of this brain specific compound was demonstrated to be extremely deficient in brain, liver and skin fibroblasts from patients who died of Krabbe's disease. This deficiency is the third enzymatic block demonstrated in this disorder. The β-galactosidase activity toward galactocerebroside and psychosine is also extremely deficient. This finding provides new information about the substrate recognition pattern of this enzyme and about the possible etiology of globoid cell leukodystrophy.  相似文献   

13.
Competition and thermal inactivation experiments with different potential natural substrates indicated that in homogenates of human fibroblasts one single enzyme is acting on both (alpha 2-3) and (alpha 2-6) sialosyl linkages of oligosaccharides and glycoproteins, but not of the ganglioside GM3. N-Acetylneuraminic and 2-deoxy-2,3-dehydro-N-acetylneuraminic acids are competitive inhibitors, whereas chondroitin 4-sulphate and the drug Suramin are potent inhibitors of undefined type.  相似文献   

14.
Galactosyltransferase, GalT-3 (UDP-Gal:GM2 beta 1-3 galactosyltransferase) has been characterized and solubilized from 19-day-old embryonic chicken brain, and purified to over 2000-fold using mixed-modal chromatography on a omega-aminohexyl Sepharose column and affinity chromatography on a UDP-hexanolamine Sepharose column. The activity of purified GalT-3 was modulated by phospholipids in vitro with stimulation observed specifically with dipalmitoyl phosphatidylethanolamine (PE). All natural phospholipids tested (PE, PC and PI) inhibited GalT-3 activity. Enzyme activity was affected by the structure of the phospholipid vesicle. It was stabilized by the hexagonal (dipalmitoyl PE) structure and inhibited by the bilayer (dielaidoyl PE) structure. The long-chain fatty acid moiety of the glycosphingolipid substrate, GM2, was found to be necessary for optimum enzyme activity. In the absence of fatty acid, the modified substrates, lyso-GM2 and acetyl-GM2, had a 10-fold increased Km and a 4-8 fold decreased Vmax compared to the normal substrate. We postulate that GalT-3 belongs to a group of glycosyltransferases having recognition for both the carbohydrate as well as the hydrophobic domains (HY-CARS) of their substrates and that the fatty acid moiety of either the substrate (GM2) or a heterotropic effector (phospholipid) plays an important role in regulating the activity of this enzyme.  相似文献   

15.
Two fluorescent derivatives of cerebroside sulfate ('sulfatide') have been synthesized and used as substrates for determining arylsulfatase A activity. These were 12-(1-pyrene)dodecanoyl cerebroside sulfate (P12-sulfatide) and 12(1-pyrenesulfonylamido)dodecanoyl cerebroside sulfate (PSA12-sulfatide). When incubated at pH 5.0 in the presence of 5 mM MnCl2 and 5.5 mM of taurodeoxycholate, either substrate was hydrolyzed by arylsulfatase A of human leukocytes. The rate of hydrolysis was proportional to the incubation time and concentration of enzyme; Michaelis-Menten type kinetics were observed with increasing concentrations of substrate. For determining the rate of hydrolysis, each of the two products (i.e., P12- and PSA12-cerebrosides) were separated from the bulk of respective unreacted sulfatide on small columns of DEAE-Sephadex A-25 and their fluorescence intensities read at 343-378 and 350-380 nm for the excitation and emission wavelengths for P12- and PSA12-cerebrosides, respectively. When extracts of skin fibroblasts derived from normal individuals and patients with Maroteaux-Lamy (lacking arylsulfatase B) or metachromatic leukodystrophy (lacking arylsulfatase A) were used as source of enzyme, P12-sulfatide was hydrolyzed by the former two but not by the latter cell extract. Several derivatives of cerebroside sulfate were also synthesized and found to inhibit the hydrolysis of pyrenesulfatide by leukocyte arylsulfatase A. The results demonstrate that these two pyrene containing sulfatides can be effectively used as specific substrates for the determination of arylsulfatase A activity in extract of cells and most probably also of tissues.  相似文献   

16.
17.
4-hydroxy-4-androstene-3,17-dione (4-OHA) has been shown to be a potent inhibitor of aromatase activity. It is effective in the control of estrogen-dependent processes in female subjects and may potentially be useful in the treatment of estrogen-dependent processes in men. Human foreskin fibroblasts grown in cell culture provide a model to investigate the effects of 4-OHA on extraglandular aromatase activity as well as the ability of the compound to influence androgen receptor binding and the 5 alpha-reduction of testosterone (T). Initial experiments were carried out to determine the potency of 4-OHA in genital skin fibroblasts by incubating cells with 4-OHA over a range of concentrations. When aromatase activity was determined at a substrate concentration close to the apparent Km of the enzyme, a 44% inhibition of enzyme activity occurred at a mean concentration of 5 nM 4-OHA. Enzyme kinetic studies analyzed by Eadie-Hofstee plots demonstrated competitive inhibition by 4-OHA with a mean apparent Ki of 2.7 nM. When 5 alpha-reductase activity was determined in the presence of 200 nM [3H]T, in the absence or presence of 4-OHA, a 50% inhibition of enzyme activity occurred at an inhibitor concentration of 3 microM. In androgen receptor binding studies, 4-OHA possessed 1% of the affinity of dihydrotestosterone (DHT) for [3H]DHT binding sites. In summary: 4-OHA is a potent and specific inhibitor of aromatase activity in human genital skin fibroblasts, the affinity of the enzyme for 4-OHA being greater than its affinity for the substrate, androstenedione. The influence of 4-OHA on 5 alpha-reductase activity and androgen receptor binding is minimal.  相似文献   

18.
A new bacterial sialidase (N-acetylneuraminate glycohydrolase, EC 3.2.1.18) isolated from the culture filtrate of Arthrobacter ureafaciens was characterized in detail with respect to its action on sialoglycolipids. Strong electrolytes had a reversible inhibitory effect on the action of the enzyme on brain gangliosides in accordance with Debye-Hückel effect of ionic environment on ionic activity, and resulted in an acidic shift and a broadening of the pH optimum. Both ionic and non-ionic detergents markedly enhanced the enzymic activity on the gangliosides, and caused an acidic shift on the pH optimum of this enzyme. Sulfhydryl groups seemed to be involved in its active site. This enzyme had a highly specific action on sialidase-resistant ganglioside GM1, showing about 100-fold higher activity on GM1 than Clostridium perfringens sialidase, the only sialidase so far reported to cleave the lipid substrate in the presence of bile salts. In the absence of detergents, the activity of A. ureafaciens sialidase on GM1 was very low. Ganglioside GM1 in either the monomeric or micelar form was hydrolyzed to asialo-GM1 by A. ureafaciens sialidase most efficiently in the presence of sodium cholate of about three times the GM1 molar concentration. The presence of detergents increased both the Km and Vmax values for ganglioside GM1. The oligosaccharide prepared from GM1 by ozonolysis was cleaved well by this sialidase in the absence of detergents, and no detergent was found to affect the hydrolysis. The Km value for the sugar substrate was about two orders of magnitude greater than that for the corresponding lipid substrate. It is suggested that the hydrophobic ceramide moiety increases affinity of the lipid substrate to the enzyme, but inhibits hydrolysis of the substrate, possibly due to its hydrophobic interaction with hydrophobic portions of the enzyme molecule (resulting in lower Km and Vmax for lipid substrates). This inhibition may be released by detergent due to formation of mixed micelles of sialoglycolipid and detergent molecules. It is also indicated that recognition of the specific saccharide structure of GM1 by individual sialidases is essential for release of the resistant sialyl residue, and that A. ureafaciens sialidase seemed to have an isoenzymic or oligomeric structure.  相似文献   

19.
The cerebro-hepato-renal (Zellweger) syndrome is an autosomal recessive disorder biochemically characterized by the absence of morphologically distinguishable peroxisomes. Key enzymes involved in the biosynthesis of ether phospholipids, i.e., dihydroxyacetone phosphate acyltransferase and alkyl dihydroxyacetone phosphate synthase, are located in mammalian (micro)peroxisomes. We have previously shown a strikingly reduced activity of dihydroxyacetone phosphate acyltransferase in liver, brain, and cultured skin fibroblasts from Zellweger patients (Schutgens et al. 1984. Biochim. Biophys. Res. Commun. 120: 179-184). We have now extended these investigations by studying alkyl dihydroxyacetone phosphate synthase in cultured human skin fibroblasts. Enzymatic activity was determined by measuring the formation of radioactive alkyl dihydroxyacetone phosphate from palmitoyl dihydroxyacetone phosphate and [1-14C]hexadecanol as substrates. The enzyme was optimally active at pH 8.5 and was stimulated (about 2-3-fold) by the presence of 0.05% (v/v) Triton X-100. The apparent KM values for the enzyme in control fibroblasts amounted to 35 microM for palmitoyl dihydroxyacetone phosphate and 90 microM for hexadecanol. The reaction became inhibited at higher concentrations of both Triton X-100 and palmitoyl dihydroxyacetone phosphate. Control skin fibroblasts showed alkyl dihydroxyacetone phosphate synthase activity of 69 +/- 28 pmol X min-1 X mg-1 (n = 7), while fibroblasts from patients had an activity of only 6.3 +/- 1.7 pmol X min-1 X mg-1 (n = 7). Alkyl dihydroxyacetone phosphate synthase was also found to be deficient in tissue homogenates of Zellweger patients. The specific activity of this enzyme in liver, kidney, and brain homogenates from Zellweger patients was less than 15% of that in the corresponding tissues from controls.  相似文献   

20.
Prolidase deficiency (PD) is a recessive disorder of the connective tissue caused by mutations in the prolidase, a specific peptidase, cleaving the dipeptides with a C-terminal prolyl and hydroxyprolyl residue. PD is a complex syndrome characterized mainly by intractable skin lesions, recurrent respiratory infections and mental retardation. The relation between prolidase biological functions and the disease is still largely unknown. We studied the effect of a prolidase inhibitor, N-benzyloxycarbonyl-l-proline (Cbz-Pro), in vitro on prolidase from human fibroblasts and in vivo on murine erythrocytes prolidase. A 90% inhibition was detected incubating cellular extracts at 1:1 ratio of Gly-Pro substrate: Cbz-Pro inhibitor. Pulse experiments performed incubating human fibroblasts with 6 mM Cbz-Pro revealed that the inhibitor uptake was completed in about 1 min. The Cbz-Pro uptake was saturable and pH dependent. Long-term incubation of fibroblasts with Cbz-Pro caused mitochondria depolarization and increased cellular death as reported for long-term culture of fibroblasts from PD patients. An inhibitory effect of Cbz-Pro has also been shown in vivo. Our results demonstrated that Cbz-Pro is a potent inhibitor of prolidase in cultured fibroblasts and it can be used in vivo to better characterize the prolidase enzyme and further investigate PD physiopathology.  相似文献   

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